There is a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence of proteins, hormones, drugs, viruses, microorganisms, narcotics and steroids must be determined rapidly and accurately for effective research, diagnosis and treatment.
A wide variety of analytical methods have been developed in recent decades to detect the noted substances. The methods have become highly reliable and in some instances, suitable for automation, as well as suitable for use in kit form. Most of such methods rely on what are known in the art as "specific binding reactions" between a substance to be detected (identified herein as a "specific binding ligand" or "ligand") and a corresponding "receptor" which recognizes and reacts with the ligand specifically. Most known specific binding reactions are between immunoreactants (in "immunoassays"), such as antibodies with antigens or antibodies with haptens, but others are also known such as avidin with biotin.
In general, immunoassays can provide a qualitative and/or quantitative determination of the presence or absence (or quantity) of a specific antigen, antibody or antigen-antibody complex. In one form of immunoassay, known as a "competitive binding immunoassay", a labeled analog of the ligand to be determined is placed in competition with a fixed amount of an appropriate antibody which can react with both the ligand and the ligand analog. The label on the analog can be appropriately detected in its "free" or complexed (that is, reacted) form. Such detection will then tell the user how much ligand is in the sample being tested.
In an alternative immunoassay format known as a "sandwich" immunoassay or immunometric assay, the ligand is contacted with two or more receptor molecules which bind to the ligand at different epitopic sites. One receptor is appropriately labeled and the other is either immobilized on a solid substrate, or is capable of being immobilized thereon. The amount of ligand is directly proportional to the amount of bound complex among the ligand and the two receptors.
Immunoassays have been traditionally carried out in solution, or in test devices where fluids are removed in some fashion from the reagents participating in the assay. Although solution techniques have enjoyed broad acceptance in this area, they typically require analyzer equipment often having intricate solution handling and transport capabilities. Moreover, the analytical equipment used in such assays can involve complex liquid handling, and may require skilled personnel, both for operation and the precise cleaning that may be needed to avoid sample to sample contamination.
An alternative to solution chemistry is the use of dry analytical elements. It should be understood that not all aqueous solutions can be employed in dry analytical elements because of interference from coating agents, binders, and other reagents necessary to maintain structural integrity in said elements. Also, a multiplicity of scientific disciplines are often required in successful element construction. Moreover, dry analytical elements must use compartmentalization to segregate incompatible components; such is not the case in solution chemistry where separate liquid storage and successive liquid additions can be employed.
Dry analytical elements and their use for immunoassays are described in numerous publications, including U.S. Pat. No. 4,258,001 to Pierce et al., U.S. Pat. No. 4,670,381 to Frickey et al., WO 82/2601 (published Aug. 5, 1982), European Patent Application No. 051 183 (published May 12, 1982) and European Patent Application No. 066 648 (published Dec. 15, 1982).
Improved dry analytical elements and their use in immunoassays are described in copending and coassigned application, U.S. Ser. No. 938,460, filed Aug. 31, 1992 to Belly et al. in which enzyme labels are utilized for detection. Horseradish peroxidase is the preferred enzyme label disclosed in the application of Belly et al. Such elements allow for the detection of analytes present in very low concentrations using a particular washing technique to separate unbound reactants from bound (or complexed) immunoreactants.
In the immunoassays carried out in the dry analytical elements using peroxidase as the label, the stability of the peroxidase is highly important since any change in its concentration critically affects assay sensitivity. In the assays described in U.S. Pat. No. 5,372,932 to Friedman et al., 4'-hydroxy or 4'-alkoxyarylacetamides are used as agents to enhance the stability of the enzyme or enzyme label in a dry analytical element. Although these agents have resulted in improved stability, it has been observed that the stability of the peroxidase label is still less than desirable in dry analytical elements.
Recently, Butler and Walker have described a family of vanadium bromoperoxidases (VBrPO) extracted from aquatic and marine algae and some from terrestrial lichens and fungi that, in the presence of hydrogen peroxide and bromide anion, catalytically produce activated bromine species which are potent oxidants. (See, Butler et al., Chem. Revs., 93, pp. 1937-1944, 1993 and references cited therein).
One of the problems with most haloperoxidases is that their pH optima are generally in the lower pH range, i.e., 3-5. This presents a problem with prior art analytical elements which depend upon a peroxy anion; peroxides typically have a pKa of about 11.5. The vanadium bromoperoxidases work effectively in a pH range of about 6-10.
Although the analytical elements and methods disclosed in the prior art show enhanced assay sensitivity and stability, there is still a need for further improvements in this field. For example, there is still a need for providing a simpler analytical element which does not require the use of stabilizing agents as described in U.S. Pat. No. 5,372,932 to Friedman et al. Furthermore, there still is a need to provide an element which has improved enzyme stability and more sensitivity than the known prior art systems.